Biopolymers are biocompatible polymers that are useful for a wide variety of biomedical applications, such as for surgical aids, to prevent or reduce the formation of surgical adhesions, and for drug delivery applications. Many biopolymers are naturally occurring substances found in the body, and therefore do not have any unacceptable toxic or injurious effects on biological function. An example of such a biopolymer is hyaluronic acid (xe2x80x9cHAxe2x80x9d), a naturally occurring mucopolysaccharide found, for example, in synovial fluid, in vitreous humor, in blood vessel walls and the umbilical cord, and in other connective tissues. Hyaluronic acid consists of alternating N-acetyl-D-glucosamine and D-glucuronic acid residues joined by alternating xcex2 1-3 glucuronidic and xcex2 1-4 glucosaminidic bonds, so that the repeating unit isxe2x80x94(1xe2x86x924)-xcex2-D-GlcA-(1xe2x86x923)-xcex2-D-GlcNAcxe2x80x94. In water, hyaluronic acid dissolves to form a highly viscous fluid. The molecular weight of hyaluronic acid isolated from natural sources generally falls within the range of 5xc3x97104 up to 1xc3x97107 daltons.
U.S. Pat. No. 4,582,865, to Balazs et al. states, inter alia, that cross-linked gels of HA can slow the release of a low molecular weight substance that is dispersed therein but not covalently attached to the gel macromolecular matrix. See, also, U.S. Pat. No. 4,636,524, which contains a disclosure of related technology. Both of these patents describe HA compositions in which the HA is crosslinked by reaction with divinyl sulfone, and the use of the crosslinked HA compositions in drug delivery applications.
R. V. Sparer et al., 1983, Chapter 6, pages 107-119, in T. J. Roseman et al., Controlled Release Delivery Systems, Marcel Dekker, Inc., New York, describes sustained release of chloramphenicol covalently attached to hyaluronic acid by an ester linkage, either directly or in an ester complex including an alanine bridge as an intermediate linking group. The HA is modified by attaching cysteine residues to the HA via amide bonds, and then the cysteine-modified HA is crosslinked by forming disulfide bonds between the attached cysteine residues. Similarly, I. Danishefsky et al., 1971, in Carbohydrate Res., Vol. 16, pages 199-205, describe the modification of a mucopolysaccharide by converting the carboxyl groups of the mucopolysaccharide into substituted amides by reacting the mucopolysaccharide with an amino acid ester in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (xe2x80x9cEDCxe2x80x9d) in aqueous solution. See, also, U.S. Pat. No. 4,937,270 and U.S. Pat. No. 5,760,220 which describe the modification of hyaluronic acid by reaction of the carboxyl groups of the biopolymer with a nucleophile to produce a water insoluble amide, and the use of those compositions for controlled release drug delivery.
A series of patents assigned to VivoRx Pharmaceuticals, Inc., describe compositions for the in vivo delivery of insoluble pharmaceutically active agents. Delivery of the drug substances is achieved, for instance, by encasing the active agent in a polymeric shell formed from a biocompatible polymer. The biocompatible polymer may be protein, lipid, DNA molecule or polysacharide, and the pharmaceutically active agent may be a therapeutic protein such as taxol. The polymer contains covalently attached sulfhydryl groups or disulfide linkages which can be crosslinked to form disulfide bonds. The polymeric shell is formed using ultrasonic irradiation techniques. These compositions are described as being less toxic, being more soluble, and having improved targeting as compared to prior art compositions. Relevant VivoRx patents include U.S. Pat. Nos. 5,498,421; 5,439,686; U.S. Pat. No. 5,362,478; U.S. Pat. No. 5,635,207; U.S. Pat. No. 5,560,933; U.S. Pat. No. 5,635,207 and U.S. Pat. No. 5,639,473.
U.S. Pat. No. 5,496,872 relates to biocompatible and biodegradable crosslinkable polymers having reactive thiol groups. The reactive thiol groups can be crosslinked to form disulfide linkages between adjacent molecules, resulting in a three dimensional network. These polymers can be used for binding tissues or binding tissues with implanted biomaterials.
U.S. Pat. No. 5,932,552 describes a keratin hydrogel having biomedical applications. The hydrogel is formed from crosslinked keratin bound by disulfide linkages. Among the biomedical applications described in the patent are uses of the hydrogels for cell scaffolding in tissue repair.
U.S. Pat. Nos. 5,354,853 and 5,451,661 describe, respectively, the preparation of phospholipid-saccharide conjugates, and lipids conjugated to biologically active agents such as peptides, proteins and nucleic acids. These conjugates are described as being particularly useful in drug delivery applications.
U.S. Pat. No. 5,902,795, to Toole et al., discloses hyaluronic acid oligosaccharides, having between one and sixteen repeating units, which are used to treat tumors in mammals. The patent states that the oligosaccharides act to reduce the level of membrane-associated hyaluronan-binding proteins, which are expressed on the surface of certain tumor cells during cell migration. The treatment is believed to reduce the incidence of tumor metastasis in the mammals.
A. Burnkop-Schnurch et al., J. Controlled Release, 2000, 66, 39, describes the synthesis of carboxymethyl cellulose (xe2x80x9cCMCxe2x80x9d) and polycarbophil modified with L-cysteine using carbodiimide chemistry. The polymers are reacted with the cysteine to form an amide bond between the primary amino group of the amino acid and the carboxylic acid of the polymer. The thiolated polymers were allowed to oxidize to form disulfide bridges. The dissolution of these tablets, both with and without drugs, was analyzed. The tablets were found to have improved stability and viscoelasticity.
Copending U.S. patent application Ser. No. 09/430,857 now abandoned relates to surfaces that have been modified by the attachment of hyaluronic acid. The surface can be part of a medical device, such as a stent or a surgical tubing. The surface is modified to include a reactive amino group that reacts with a derivatized hyaluronic acid. The modified devices and instruments are hydrophilic, and have anti-fouling and anti-platelet adhesion characteristics, thereby producing a reduction in risks associated with thrombosis.
The conjugated biopolymers of this invention represent a significant improvement over drug delivery vehicles of the prior art due, in part, to the site-specific reaction between the biopolymer and the therapeutic agent which increases the stability and activity of the therapeutic agent upon delivery to the desired site within a subject.
The present invention features a biopolymer-therapeutic agent conjugate in which the biopolymer and therapeutic agent are joined by a disulfide bond. The biologically active conjugate of this invention is useful as a drug delivery vehicle for the in vivo delivery of the therapeutic proteins to specific cells, organs or tissues in a subject. Drug delivery specificity is achieved by appropriate selection of the structure and molecular weight of the biopolymer.
The chemistry used to prepare the conjugates permits the site-specific reaction between the biopolymer and the therapeutic agent. The therapeutic agent contains a reactive thiol group, which can be present in an unmodified version of the therapeutic agent, as in the case of cysteine for example. Alternatively, the thiol group can be introduced into a modified version of a therapeutic agent that does not normally contain a reactive thiol group.
In one embodiment, the therapeutic agent can be reacted, through the reactive thiol group, with a chemically modified version of the biopolymer. This reaction typically occurs at a pH in the range of from about 6.0 to about 10. The biopolymer is activated and modified by reaction with an activating agent, such as a carbodiimide, and reacted with an organic disulfide compound. The organic disulfide compound contains a terminal group, such as an amino group or a hydroxyl group, which is reactive with the carboxylic acid group of the biopolymer in the presence of the activating agent. The reaction of the biopolymer, activating agent and organic disulfide compound occurs at a pH of from about 2.0 to 8.0.
In another embodiment, the therapeutic agent can be reacted, again through the thiol group, with the reducing end of the biopolymer. The biopolymer is first reacted with an organic disulfide compound containing a terminal group, such as an amino group or a hydroxyl group, which is reactive with the terminal carboxyl group of the biopolymer. The reaction of the biopolymer and organic disulfide compound occurs over a wide pH range, typically at a pH of from about 2.0 to 9.0.
In one aspect, the reaction of the biopolymer and therapeutic agent results in the attachment of the biopolymer to the therapeutic agent through a disulfide bond. The linking group or spacer, which can be a lower alkyl, separates the biopolymer from the therapeutic agent. The linking or spacer is a residue resulting from the cleavage of the organic disulfide compound by the reactive thiol of the therapeutic agent.
Typical biopolymers include any of the polyanionic polysaccharides, such as hyaluronic acid and any of its hyaluronate salts, such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate, carboxymethyl cellulose, carboxymethyl amylose, chondroitin-6-sulfate, dermatin sulfate, heparin, and heparin sulfate, as well as polyacrylic acid, polycarbophil, carboxymethyl chitosan, poly-xcex1-glutamic acid, poly-xcex3-glutamic acid, carrageenan, and sodium alginate. The common feature of the biopolymers of this invention is that they are biocompatible, as that term is defined herein, they contain carboxylic acid functionality, and they can be modified to react with an organic disulfide compound. Such modification can occur, for instance, by reaction of the biopolymer with a suitable activating agent, such as a carbodiimide, to render the carboxylic group vulnerable to nucleophilic attack by, for instance, an amine or a hydroxyl. Alternatively, the modification can occur at the terminal or end group of the biopolymer by reduction of a terminal carbonyl group using a Schiff base.
In a preferred embodiment, the biopolymer is hyaluronic acid having a molecular weight in the range of from about 7.5xc3x97102 daltons to about 1xc3x97107 daltons. The hyaluronic acid is preferably activated by reaction with an activating agent to render it vulnerable to nucleophilic attack. Suitable activating agents for this purpose include carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide.
The organic disulfide compound can be virtually any organic compound having a disulfide bond. Preferably, the disulfide bond is positioned at one end of an alkyl chain, while the other end of the chain terminates in a group reactive with the carbonyl group of the biopolymer. Preferably, the group that reacts with the biopolymer is an amino, carboxyl or hydroxyl group, but most preferably an amino group. In addition to being capable of reacting with the biopolymer, the organic disulfide compound is also capable of reacting with the active thiol group of the therapeutic agent. Preferred organic disulfide compounds include, in general, the nitro-pyridines, thio-pyridines, substituted S-phenyl disulphides, S-sulfonate derivatives, 9-anthrymethyl thioesters, S-carboxymethyl derivatives and nitro-thiobenzoic acid derivatives. More preferably, the organic disulfide compound is a thio-nitro-pyridine, and most preferably 3-nitro-2-pyridinesulfenyl-ethylamine.
The therapeutic agent is preferably one or more of the following: small organic molecules, proteins, nucleic acids, antibodies, peptides, amino acids, lipids, polysaccharides, cell growth factors, and enzymes. More preferably, the therapeutic agent is native or recombinant colony-stimulating factor (xe2x80x9cCSFxe2x80x9d), an amino acid or glucocerebrosidase. The therapeutic agent should contain a reactive thiol group to react with the modified biopolymer. The reactive thiol group can either be inherently part of the therapeutic agent, as in the case of cysteine, or the reactive thiol group can be introduced into the therapeutic molecule using known techniques. For example, a free thiol group can be introduced into a recombinant therapeutic protein molecule for conjugation and modification. Furthermore, some therapeutic drugs, such as Captoprilxe2x80x94a drug used to treat hypertensionxe2x80x94inherently contain a free sulfhydryl group as shown in the structure below: 
The amino groups of therapeutic agents can be conveniently converted into thiols by reaction with Traut""s Reagent (aminothiolane).
The therapeutic agent is selected for the particular indication that is to be treated, and the biopolymer is selected, both as to its type and molecular weight, for its ability to target a particular organ, cell or tissue. For instance, a therapeutic agent for treating Gaucher""s Disease, a serious liver ailment, is the enzyme glucocerebrosidase. Glucocerebrosidase can be targeted to the liver by forming a conjugate with an appropriately sized hyaluronic acid molecule.
The biologically active conjugate of the present invention provides for improved stability of the therapeutic agent as compared to the use of the unconjugated or unmodified therapeutic agent, or the use of other carriers or conjugated compounds, such as polyethylene glycol (xe2x80x9cPEGxe2x80x9d) or lipids. The improved stability results in increased residence time in the body of a subject and increased circulation time in the blood stream. The conjugates of this invention also display improved targeting to specific tissues, organs and cells. Improved targeting is achieved through the selection of specific types and molecular weights of the biopolymers.
In a further aspect, the invention involves the attachment of a biopolymer onto the surface of a substrate by means of a disulfide linkage. The substrate can be a polymeric material, a ceramic or a metal. Preferably, the substrate is part of a medical device or instrument, such as a stent, graft, suture, catheter, tubing or guidewire. The substrate is modified to contain an amino group, which can then be converted into a thiol group. The substrate can then be reacted with the biopolymer modified with the organic disulfide compound to immobilize the biopolymer onto the substrate.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein, including published patent applications, and issued or granted patents, are hereby incorporated by reference in their entireties. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.